Hemoglobin (Hb) is the major constituent of the erythrocyte which carries oxygen from the lungs throughout the body. When contained in red blood cells, Hb exists as a tetramer structure composed of two oxygen linked αβ dimers each having a molecular weight of about 32 Kd. Each α and β subunit of each dimer has a protein chain and a heme molecule. The sequences of the α and β protein chains are known. Hb is a potentially useful blood substitute for transfusions, and has been proposed as a reagent to trap nitric oxide in septic shocks, and to modulate tissue oxygenation during the radiation therapy of cancer. Recombinant DNA technology also has afforded the generation of modified Hb with oxygen affinities modulated for special needs of individual therapeutic applications.
Vasoactivity of acellular Hb, i.e. the constriction of arterioles and capillaries, when infused with purified acellular Hb solutions, or intra molecularly crosslinked Hbs, has been the major impediment for developing Hb-based oxygen carriers (Savitzsky et al. 1978, Sloan et al. 1999, Saxena et al. 1999). The vasoactivity has been attributed to the NO scavenging effect of Hb (Doharty et al. 1998). Two molecular approaches, that are very distinct from one another, have been advanced to in an attempt to overcome NO scavenging activity of Hb. The first approach is the recombinant DNA approach, which has attempted to reduce the nitric oxide scavenging activity of Hb by modifying the NO binding activity of Hb by site-specific mutagenesis of the distal heme pocket (Eich et al, 1996). The second approach is the chemical approach, in which the molecular size of Hb is enhanced through oligomerization, which will reduce or possibly completely inhibit the extravasation of Hb from the vascular space into the interstitial space (Hess et al. 1978, Thomas et al. 1993, Muldoon et al. 1996, Macdonal et al. 1994, Furchgott 1984, Kilbourn et al. 1994). However, the size-enhancing approach will be successful only if the vasoactivity of Hb is essentially mediated as a result of extravasation. Though the oligomerization mediated size enhancement of Hb has shown some reduction in the vasoactivity of Hb, a non-hypertensive Hb solution has not been generated by either recombinant DNA technology or by the size enhancement approach that involved the oligomerization of Hb using small molecular bifunctional reagent. One exception is the oligomerized product of Hb (Matheson et al. 2002), that has a molecular size far in excess of 300 kDa and with an average molecular radius of 24 nm, and was found to be non-hypertensive and found not to extravasate. However, most of the current oligomerized products that are in clinical trials have a molecular weight in the range of 200 to 250 kDa.
The demonstration that Enzon polyethylene glycolylated (PEGylated) Hb, that carries ten copies of PEG-5000 chains linked to Hb at its α and ε-amino groups is non-hypertensive has stimulated the research in the blood substitute field (Rolfs et al. 1998). The NO binding activity of intra-tetramerically crosslinked Hbs, oligomerized Hbs and PEGylated Hbs (Winslow et al, 1998, Vandegriff et al, 1997) do not show a direct correlation with their ‘pressor effects’. Thus, the reduction in the ‘pressor activity’ of acellular Hb does not appear to be a direct correlate of, either the NO binding activity of the preparation or of the molecular size of the preparation. But the PEGylated Hbs exhibited considerably lower level of vasoactivity as compared to the oligomerized Hb. The PEG-Bv-Hb of Enzon that carries 10 copies of PEG-5,000 exhibited hardly any ‘pressor effect’. Vandegriff et al (1998) have noted that PEG-Bv-Hb exhibited high viscosity and oncotic pressure as compared to that of oligomerized samples of Hb. The molecular radius of Enzon PEGylated Bv-Hb calculated from the oncotic pressure was considerably larger (15 nm) than that of oligomerized Hbs and the molecular radius calculated is not consistent with its calculated molecular mass of 114,000 daltons (Vandegriff et. al. 1998). Accordingly, it has been hypothesized that size of Hb should be increased to a molecular radius of around 15 nm, and this should be accompanied by considerable increase in the viscosity and oncotic pressure to generate a non-hypertensive Hb solution (Winslow 1999).
In the non-hypertensive Enzon Pegylated Hb, PEG-5000 chains are linked to the α and ε-amino groups of bovine Hb PEG-chains by isopeptide bonds. The covalent attachment of PEG is accompanied by the loss of the net positive charge of the amino groups derivatized. In a recent study, it was demonstrated that monofunctional modification of rHb1.1 with glutaraldehyde, lowers the vasoactivity of Hb to some degree, though the oligomerization of rHb1.1 reduces the vasoactivity of Hb to a higher degree. Thus in understanding the molecular basis of neutralization of the vasoactivity of Hb by PEGylaltion, the potential role of the modification of the surface charge of Hb that accompanies the PEGylation of HB needs to be considered.
To expose the correlation between the perturbation of the surface charge of Hb resulting from PEGyaltion with the generation of non-hypertensive Hb, new approaches have been developed relating to the conservation PEGylation of Hb, i.e. PEG-modification of Hb without altering the surface charge of Hb (Acharya et al. 1996). The high reactivity and selectivity of PEG-maleimide to Cys-93(β) of Hb under oxy conditions has been used to prepare homogeneous PEGylated Hb carrying two copies PEG-chains per tetramer. Three different preparations of PEG-HbA carrying two copies each of PEG-5K, or PEG-10K or PEG-20K have been generated. The changes in the molecular volume (hydrodynamic volume), molecular radius, viscosity, and oncotic pressure of Hb has been correlated with the mass of the PEG covalently linked to Hb; and all of these molecular properties have been correlated with pressor effect. Though the viscosity and the oncotic pressure of (PEG20K)2-Hb is comparable to that of Enzon PEG-Bv-Hb, a non-hypertensive Hb molecule, this PEGylated Hb was vasoactive. Thus, the solution to vasoactivity problem cannot be achieved by simply endowing the molecule with an increase in viscosity, oncotic pressure and the molecular volume (hydrodynamic volume).